Bean Extract-Based Gargle for Efficient Diagnosis of Active COVID-19 Infection Using Rapid Antigen Tests

ABSTRACT The antigen-based rapid diagnostic test (Ag-RDT) using saliva specimens is fast, noninvasive, and suitable for SARS-CoV-2 self-testing, unlike nasopharyngeal swab (NPS) testing. We evaluated a novel Beanguard gargle (BG)-based virus collection method that can be applied to Ag-RDT as an alternative to the current RT-PCR with an NPS for early diagnosis of COVID-19. This clinical trial comprised 102 COVID-19-positive patients hospitalized after a governmental screening process and 100 healthy individuals. Paired NPS and BG-based saliva specimens from COVID-19 patients and healthy individuals were analyzed using NPS-RT-PCR, BG-RT-PCR, and BG-Ag-RDTs, whose diagnostic performance for detecting SARS-CoV-2 was compared. BG-Ag-RDTs showed high sensitivity (97.8%) and specificity (100%) in 45 patients within 6 days of illness and detected all cases of SARS-CoV-2 Alpha and Delta variants. In 11 asymptomatic active COVID-19 cases, both BG-Ag-RDTs and BG-RT-PCR showed sensitivities and specificities of 100%. Sensitivities of BG-Ag-RDT and BG-RT-PCR toward salivary viral detection were highly concordant, with no discrimination between symptomatic (97.0%), asymptomatic (100%), or SARS-CoV-2 variant (100%) cases. The intermolecular interactions between SARS-CoV-2 spike proteins and truncated canavalin, an active ingredient from the bean extract (BE), were observed in terms of physicochemical properties. The detachment of the SARS-CoV-2 receptor-binding domain from hACE2 increased as the BE concentration increased, allowing the release of the virus from hACE2 for early diagnosis. Using BG-based saliva specimens remarkably enhances the Ag-RDT diagnostic performance as an alternative to NPS and enables noninvasive, rapid, and accurate COVID-19 self-testing and mass screening, supporting efficient COVID-19 management. IMPORTANCE An Ag-RDT is less likely to be accepted as an initial test method for early diagnosis owing to its low sensitivity. However, our self-collection method, Ag-RDT using BG-based saliva specimens, showed significantly enhanced detection sensitivity and specificity toward SARS-CoV-2 including the Alpha and Delta variants in all patients tested within 6 days of illness. The method represents an attractive alternative to nasopharyngeal swabs for the early diagnosis of symptomatic and asymptomatic COVID-19 cases. The evidence suggests that the method could have a potential for mass screening and monitoring of COVID-19 cases.

(b) Agree to participate in this trial and provide written informed consent.
(c) Permit the investigator to access the patient's medical records relevant to study procedures.
(d) Provide paired NPS and BG-based saliva specimen. Medical professionals collected NPS specimens from participants by having them spit into a tube after swirling and gargling 5 mL of BG for 2 min.
Exclusion criteria (a) Healthy subjects with a body temperature higher than 37.0 ºC (b) Healthy subjects with respiratory symptoms (c) Not willing to provide sufficient BG-based saliva specimen.
(d) Not willing to collect NPS specimen. . The SARS-CoV-2 spike (S) protein S1 was obtained from ACROBiosystems (Delaware, USA). Recombinant S2 was expressed and purified as described below. The concentrations of S1 and S2 were determined based on the UV absorbance at 280 nm with molar extinction coefficients of 91,775 and 42,455/ M cm, respectively, and were calculated using the ExPASy ProtParam tool (1).

Cryo-electron microscopy (cryo-EM)
The purified whole virus, SARS-CoV-2, and a mixture of SARS-CoV-2 and bean extract (BE) samples were inactivated with 2% paraformaldehyde overnight at 4 °C. Four microliters of each sample was applied to R1.2/1.3 Quantifoil holey carbon EM grids (200 mesh), which were glow-discharged for 60 s at 20 mA. The grids were then plunge-frozen in liquid ethane using a Vitrobot Mark IV (ThermoFisher Scientific) with 5 s of blotting in 100% humidity at 4 °C. Cryo-EM images were acquired using a Titan Krios (ThermoFisher Scientific), operated at 300 kV, and equipped with a Falcon III direct detector.

Enzyme-linked immunosorbent assay (ELISA) to test inhibition of RBD binding to hACE2 receptors
To assess the dissociation activity of the receptor binding domain (RBD)-human version of angiotensinconverting enzyme 2 (hACE2) complex by BE, COVID-19 Neutralizing Antibody ELISA Kit (Abnova, Taiwan) was used according to the manufacturer's instructions, with slight modifications. Briefly, the hACE2 protein was coated on 96-well ELISA plates at 4 °C overnight and unbound proteins were removed with washing buffer, followed by blocking for 1 h at 37 °C. For pre-treatment, RBD and BE solutions were incubated for 2 h at 37 °C before reaction with hACE2-coated plate. For post-treatment, COVID-19 Spike RBD Rabbit Fc-Tag protein was added to the plates and incubated for 2 h at 37 °C, followed by washing two times with each successive 2-fold dilution of BE solution. To detect bound RBD-Fc proteins in the plate, it was washed with a wash buffer provided in the ELISA kit, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody was added to each well and the plate was incubated for 1 h at 37 °C. The plate was then washed with wash buffer and subsequently treated with 3,3ʹ,5,5ʹ-tetramethylbenzidine (TMB). Color development was terminated using the stop solution in the ELISA kit. Absorbance at 450 nm was measured using a microplate reader (Bio Tek Co., USA). The percentage of RBD-hACE2 binding was calculated by measuring the differences in the amount of labeled RBD between the test and control samples.

Preparation and purification of TCan
For the preparation of truncated canavalin (TCan), sword bean (Canavalia gladiata) was proteolyzed by Bacillus subtilis at 33 °C for 4 days under conditions of dissolved oxygen (20-40% saturation) and pH 7.0. After proteolysis, the product was centrifuged (12,000 × g) at 4 °C for 15 min, and supernatant was collected and lyophilized. The final purification was performed by size exclusion chromatography (SEC) using a WTC-050S5 column (Wyatt Technology, Santa Barbara, CA) that was equilibrated with PBS (140 mM NaCl, 2.7 mM KCl, 6.5 mM Na2HPO 4 , 1.5 mM KH2PO 4 , pH 7.4). Pure protein was frozen in liquid nitrogen and stored at −80 °C. The SEC profile of TCan is shown in S5 Figure. On-line top-down analysis of TCan using UPLC-ESI-QTOF (top-down) On-line top-down MS was carried out on a waters ACQUITY UPLC I-Class system coupled to Synapt G2-S QTOF mass spectrometer (Waters Corp., USA). Briefly, 2 μL of purified TCan was injected into a 2.1 × 50 mm ACQUITY UPLC BEH300 C4 column (particle size 1.7 µm). The gradient was delivered at 400 μL/min, starting at 5% buffer B (0.1% formic acid in acetonitrile), then increased to 95% B at 6.7 min, and 5% B at 9.0 min. The mass spectrometer was operated in the ESI-Positive MS E continuum data acquisition mode with the following conditions: capillary voltage 3.0 kV, cone voltage 50 V, desolvation gas 800 L/h, source temperature 120 °C, and desolvation temperature 300 °C. Data were collected at 1 spectra/second from 500-4,000 m/z. A maximum entropy algorithm (MaxEnt1) was used for mass deconvolution. Deconvolution was performed with a range of 1,500-2,600 Da around the expected mass, a target resolution of 0.5 Da, with 100 iterations of MaxEnt1. The electrospray spectra of TCan is shown in Supplementary S6 Figure. Protein analysis using Nano UPLC-QTOF for TCan analysis (bottom-up) The purified TCan was digested using the "Tube-Gel digestion" protocol as described previously (2). LC-MS/MS was conducted according to a previous procedure (3). The tryptic peptide mixtures were dissolved with 0·5% trifluoroacetic acid prior to further analysis. A 5 μL dissolved sample was added onto a 100 μm × 2 cm nanoviper trap column and 15 cm × 75 μm nanoviper analysis column (Thermo Fisher Scientific) at a flow rate of 300 nL/min and were eluted with a gradient of 5%-40% acetonitrile over 95 min. All MS and MS/MS spectra captured by the Q Exative Plus mass spectrometer (Thermo Fisher Scientific) were acquired in the data-dependent top 12 mode. The MS/MS data were analyzed using MASCOT 2·7, with a parameter corresponding to a false discovery rate (FDR) of 1%. One missed cleavage was allowed for identifying the peptides from TCan.

N-Terminal sequencing of TCan
For N-terminal amino acid sequencing, the protein samples were transferred from an SDS-PAGE gel to a PVDF membrane and the transferred protein band on the membrane was analyzed using the LC 492 Protein Sequencing System (Applied Biosystems Instruments, USA).

Preparation of SARS-CoV-2 spike protein
The gene encoding S2 of the SARS-CoV-2 spike protein (accession number QKU5385.1, amino acid residues 686-1213) was codon-optimized for protein expression in Nicotiana benthamiana, synthesized by GenScript Biotechnology Corp. (Nanjing, China) and cloned into a pCAMBIA1300 vector with an 8 histidine tag, an ER retention signal HDEL, and a Cor1 sequence. N. benthamiana leaves (fresh weight) expressing recombinant S2 were harvested and homogenized using a blender in the presence of an extraction buffer (25 mM Tris-HCl [pH 8.0], 300 mM NaCl, 10 mM imidazole, 5% glycerol, 1% Sarkosyl, 10 mM H2O2, and 1 mM PMSF). To remove debris, the extracts were centrifuged at 20,000 g for 40 min, and supernatants were filtered through Miracloth (EMD Millipore Corp., Billerica, MA, USA). The resulting supernatant was loaded onto a 100-mL Ni-IDA agarose column (Bio-Rad, Seoul, Republic of Korea). The column was washed with a washing buffer consisting of 25 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 10 mM imidazole. S2 was eluted with the same buffer containing 30-300 mM imidazole. The proteins were further purified using gel-filtration chromatography (Superdex 200 Increase 10/300 GL, GE Healthcare, USA), pre-equilibrated with phosphate-buffered saline (PBS), and stored at -80 °C.
Circular dichroism spectroscopy Circular dichroism (CD) measurements were performed on a JASCO J-1500 spectrophotometer (JASCO, Japan) using a quartz cuvette with 0·1-cm path length. Samples were prepared using 0.25 mg/mL of TCan in 20 mM sodium phosphate buffer (pH 7·5). Far-UV CD spectra from 195 to 250 nm were obtained at a scanning rate of 200 nm/min. CD signals were expressed as mean residue ellipticity [θ] (degrees cm 2 /dmol) (4). For the thermal scans, far-UV CD spectra were acquired every 5 °C from 5 to 100 °C at a heating rate of 1 °C min -1 . Then, monitoring CD signals at 204 nm was carried out by decreasing the temperature from 100 to 5 °C at a rate of 10 °C/ /min. The temperature was controlled using an RW-0525G low-temperature bath circulator (Lab Companion, Republic of Korea). The melting temperature ( CD Tm), enthalpy change ( CD ΔH), and heat capacity change (ΔCp) for the heat denaturation of folded TCan were obtained by regression analysis as follows (5): where θ is the signal intensity monitored by CD. The pre-and post-unfolding baselines are described by a + bT and c + dT, respectively. T is the temperature in Kelvin, and R indicates the gas constant. CD Tm is the midpoint temperature of the thermal denaturation of TCan, ΔCp is the heat capacity change between pre-and post-unfolded TCan, and CD ΔH ( CD Tm) is the enthalpy change at CD Tm. CD spectra were analyzed using the BeStSel algorithm (6) to estimate the content of the secondary structure. The concentration of TCan was determined using the UV absorbance and a molar extinction coefficient of 8,940/M cm at 280 nm.

Dynamic and light scattering measurement
Dynamic light scattering (DLS) and static light scattering measurements of TCan in 20 mM sodium phosphate buffer (pH 7.5) were performed using a DynaPro Plate Reader II (Wyatt Technology Corp., USA) and a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, USA) at 25 °C, respectively. For DLS measurements, sample solutions containing 11·7 µM TCan before and after heat treatment were subjected to centrifugation at 10,000 rpm for 5 min, and the supernatants were loaded into a 384-well plate. The average hydrodynamic radius (Rh) of the 20 measurements was obtained. Experimental data were processed and analyzed using the DYNAMICS software (Wyatt Technology Corp., ver. 7.0). The emission spectrum of light scattering of approximately 12 µM TCan was recorded from 300 to 400 nm with an excitation wavelength of 350 nm. The slit widths of the excitation and emission wavelengths were 10 and 5 nm, respectively. Scattering of 20 mM sodium phosphate buffer (pH 7.5) was also performed as a control using the same method.
Intrinsic and extrinsic fluorescence spectroscopy All fluorescence measurements were carried out in the absence and presence of 6.0 M Urea using a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, USA) and approximately 11.7 µM TCan in 20 mM sodium phosphate buffer (pH 7·5) at 25 °C. Intrinsic fluorescence spectra were obtained by exciting phenylalanine and tyrosine/tryptophan at 240 and 280 nm, respectively. An excitation slit of 10 nm and an emission slit of 20 nm were used. ANS (50 µM) was utilized as an extrinsic fluorophore, and its emission spectrum was recorded from 370 to 600 nm with an excitation wavelength of 370 nm. The concentration of ANS was determined using UV absorbance at 350 nm and a molar extinction coefficient of 4,950/M cm. The emission spectra were monitored from 370 nm to 600 nm. The excitation and emission slit widths for intrinsic and extrinsic fluorescence were both set to 20 nm. Fluorescence measurements of 20 mM sodium phosphate buffer (pH 7·5) were carried out as a control using the same method.
Differential scanning calorimetry Differential scanning calorimetry (DSC) experiments of 17.6 µM TCan in 20 mM sodium phosphate buffer (pH 7·5) were carried out using a PEAQ-DSC instrument (Malvern Panalytical, UK). The TCan solution was heated from 20 °C to 100 °C at a heating rate of 1 °C/min. After subtraction of the buffer baseline and normalization with protein concentrations, fitting analyses were performed based on the non-two-state transition using PEAQ-DSC analysis software (Malvern Panalytical, UK). The fit equation used was based on the Levenberg-Marquardt nonlinear least-squares method (7).
where Tm is the thermal midpoint of a transition, as described above. The change in calorimetric enthalpy ( DSC ΔHcal) was determined using the DSC peak area, and the enthalpy change was based on the van't Hoff method ( DSC ∆HVH) and was estimated by exploiting the shape of the DSC peak, that is, the slope of the transition curve between folded and unfolded proteins. Cp(T) was calculated at any temperature T in Kelvin. K (T) indicates the equilibrium constant as a function of the temperature.

Isothermal titration calorimetry
Isothermal titration calorimetry (ITC) was performed using a MicroCal PEAQ-ITC (Malvern Panalytical, UK) at 37 °C. All samples, TCan, S1, and S2, were prepared in 20 mM sodium phosphate buffer (pH 7·5) containing 100 mM NaCl. TCan (287.6 µM) in an ITC syringe was titrated to solutions containing either S1 or S2 at 17 µM in an ITC cell with continuous stirring at 750 rpm. The total number of titrations was 19 with 150-s intervals between titrations. The initial delay and reference power were set to 60 s and 10 µcal/s, respectively. The dilution heat of TCan was also measured using the same method and the ITC parameters. Binding isotherms after the subtraction of dilution heat and the baseline correction were fitted to a one-set of sites binding model (8) where Q is the total heat constant, and n indicates the number of TCan molecules which bind to one of the S proteins (i.e., S1 and S2). ITC ΔHbind is the change in enthalpy for the intermolecular interactions. V0 is an active cell volume which considers the volume of the ITC cell and the volume increased by titration. The total concentrations of S proteins and TCan are displayed as [S protein]t and [TCan]t, respectively, at any given time (t). The dissociation constant is denoted as Kd. In binding isotherms of TCan-S1 and TCan-S2, a best fit was obtained without fixation of variable, and fit by fixing a value of Kd was also performed.
Size exclusion chromatography-combined multi-angle light scattering measurement Size exclusion chromatography with inline multi-angle light scattering (SEC-MALS) measurements were conducted using an instrument from Wyatt Technology (USA). Freshly purified 94·4 µM TCan solution was loaded onto a WTC-050S5 column (Wyatt Technology Corp., USA) with PBS running buffer. The flow rate was set to 0·5 mL/min. MALS and UV spectra were recorded using a DAWN HELEOS-II (Wyatt Technology Corp., USA), and Optilab T-rEX (Wyatt Technology Corp., USA) was used to determine the differential refractive index. SEC-MALS results were analyzed using Astra 6·1 software (Wyatt Technologies Corp., USA). A differential refractive index increment (dn/dc) value of 0.185 was used.

Cell culture, viruses, and treatment
To compare the cytotoxicity of BE on different cell types, two human hepatocellular carcinoma cell lines (HepG2 and Huh7 cells) and two lung origin cell lines (adenocarcinomic human alveolar basal epithelial cells, A549, and alveolar macrophages, MH-S), were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). A549 cells were cultured in RPMI 1640 containing 5% FBS and 1% streptomycin/penicillin at 37 °C in an atmosphere containing 5% CO2. MH-S cells were cultured in RPMI 1640 containing 10% FBS and 1% streptomycin/penicillin at 37 °C in an atmosphere containing 5% CO2. HepG2 and Huh7 cells were cultured in DMEM containing 10% FBS and 1% streptomycin/penicillin at 37 °C in an atmosphere containing 5% CO2. Using sterile 24-well culture plates, cells were seeded at a concentration of 2 × 10 5 in 500 μL of each medium and treated with Con A or BE at 5, 10, 25, 50, 100, 200, and 400 ppm.

Determination of cell viability
After 6 h of BE treatment, A549, HepG2, MH-S, and Huh7 cells were harvested using 0.25% trypsin-EDTA and counted using a NucleoCounter (NC-250, ChemoMetec, Gydevang, Denmark), according to the manufacturer's protocol. Cell viability was calculated as the ratio of live cells to total cells. Inhibitory concentration (IC) was also calculated from nonlinear regression of concentration-response inhibition curves using GraphPad Prism v·7 (GraphPad Software, San Diego, CA).
Measurement of reactive oxygen species (ROS) levels A549, HepG2, MH-S, and Huh7 cells were seeded and treated with BE, as previously described. The treated cells were incubated for 30 min at 37 °C in culture medium containing 3.3 μmol/L DCF-DA for ROS detection, and the cells were washed with PBS. DCF-DA intensity in the cells was immediately measured using flow cytometry (CytoFLEX, Beckman Coulter, Brea, CA, USA). ROS production in the cells is represented as a percentage of DCF-DA intensity relative to the naïve control, which was defined as 100%.
Primary splenocyte preparation and culture Primary splenocytes were isolated from BALB/c and C57BL/6 mice. Single cell suspensions were obtained by mincing the spleen and gently pressing the fragments through a 45 μm nylon cell strainer (BD Falcon, Bedford, MA, USA). The suspension was mixed with 1× RBC lysis buffer (eBioscience Inc., San Diego, CA, USA) for 5 min at room temperature. For primary culture, the spleen cells were resuspended in RPMI 1640 containing 5% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Using sterile 24-well culture plates, cells were seeded at a density of 3 × 10 6 cells in 500 μL of medium. Spleen cells were cultured for 24 or 48 h in the presence of 2.5 μg/mL Con A in a humidified atmosphere of 5% CO2 and 95% air. Cellular morphology was observed under a light microscope (20×; Leica Microsystems, Wetzlar, Germany). Cells were harvested and centrifuged at 2,000 × g for 5 min; thereafter, the supernatants were collected and stored at −70 °C until measurement of inflammatory cytokine levels by ELISA.

Experimental design for in vivo study
The following three studies were conducted to compare the toxic effects of BE: Study 1) Effect of high-dose BE acute toxicity on survival rate Study 2) Comparison of BE hepatotoxicity Study 3) Comparison of BE respiratory toxicity to determine the possibility of intratracheal instillation as a mode of administration to measure the antiviral efficacy of BE Study 1: To examine and compare the effect of BE on survival rate, seven groups were created (n = 3 per group), i.e., vehicle control group, Con A treatment groups (40, 80, and 160 mg/kg), and BE treatment groups (75, 150, and 200 mg/kg). BE was diluted in saline to create equivalent doses, which were intratracheally administered using a modified automatic video instillator (Doobae System, Seoul, South Korea). Following Con A and BE administration, the survival rate was monitored for 24 h. Study 2: To examine and compare the hepatotoxicity of Con A and BE, mice were administered Con A and BE intravenously (5 and 15 mg/kg) and intratracheally (10, 20, and 40 mg/kg). Several parameters were then evaluated, including serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), and γglutamyl transferase (GGT) levels in the blood, as well as histological changes in the liver. Study 3: To examine and compare the toxicity of Con A and BE on the lungs, mice were intratracheally administered different concentrations of Con A or BE, i.e., 1, 2.5, 5, and 10 mg/kg. Organ weight, cellular changes, and protein levels of inflammatory cytokines in bronchoalveolar lavage fluid (BALF), as well as histological changes in the lung and spleen tissues were assessed 24 h after the single administration of Con A or BE.

Intratracheal and intravenous instillation
For intratracheal instillation, mice were anesthetized by inhalation. Prior to instillation, isoflurane was delivered into the induction chamber using a small animal porTable anesthesia system (L-PAS-02, LMSKOREA, Inc., Seongnam, South Korea) equipped with an isoflurane vaporizer. The mice were then exposed to 2·5% isoflurane delivered in O2 (2 L/min) within the induction chamber until a sleep-like state was reached. Mice receiving isoflurane anesthesia were removed from the induction chamber, and instillation was performed immediately. For intravenous instillation, mice were placed in a murine restraining device. The tail vein was dilated by soaking the tail in warm (e.g., at 30-35 °C) water for 1 to 2 min. After swabbing with 70% alcohol, the tail veins were injected with the designated concentration. Mice were subsequently transferred to their cages.

Bronchoalveolar lavage fluid preparation
Twenty-four hours after Con A and BE instillation, mice were anesthetized and the left lung was ligated, while the right lungs were gently lavaged three times via a tracheal tube with a total volume of 0.7 mL PBS. The total number of cells in the collected bronchoalveolar lavage fluid (BALF) was counted using a NucleoCounter. For differential cell counts, BALF cell smears were prepared using Cytospin (Thermo Fisher Scientific) and stained with the Diff-Quik solution (Dade Diagnostics, Aguada, Puerto Rico), followed by counting of different cell types (n = 200/slide). BALF was immediately centrifuged at 2,000 × g for 5 min, and the collected supernatant was stored at −70 °C until measurement of cytokine levels by ELISA.

Histological analysis
Twenty-four hours after BE instillation, the mice were euthanized for histological analysis. Lung and liver tissues were removed and fixed in 10% (v/v) neutral-buffered formalin, dehydrated, embedded in paraffin, and cut into 4 μm sections. They were then deparaffinized with xylene and stained with hematoxylin and eosin (Sigma-Aldrich). Stained sections were analyzed using a light microscope (Axio Imager M1; Carl Zeiss, Oberkochen, Germany). The degree of inflammation was scored on a scale of 0-4.

S4 text. Supplementary Study Results and Discussion
Characterization of TCan In this work, fermented sword bean (Canavalia gladiata) was used and the active ingredient TCan was successfully produced in culture medium during the cultivation. Interestingly, N-terminal sequence of the canavalin was determined by Edman degradation as 242 LSSQDKPFN 251 , indicating that in C. gladiata truncation occurs at the N-terminal region of canavalin. To determine the mass value of the whole protein, the multiplecharge electrospray mass spectrum (S7 Fig) of TCan protein was deconvoluted to calculate protein molecular weight by MaxEnt1 software. The mass value of the TCan protein was observed to be 21,176 Da, which was predicted to be due to truncation of not only the N-terminal region but also the C-terminal lysine residue (190). The peptides of the protein were identified using Q Exative Plus mass spectrometer. A protein identification search revealed that the peptides present in the predicted TCan matched with those of canavalin (C. gladiata, P10562·1), providing 97% sequence coverage for the predicted TCan amino acid sequence using LC-MS/MS bottom-up analysis. In future, it is necessary to verify the biochemical characterization of TCan by designing a recombinant protein.

Structural characterization of TCan
To understand the molecular properties of TCan, we first performed a structural investigation using circular dichroism (CD) (S8A Fig) and fluorescence spectroscopic techniques (S8B- D Fig). Far-UV CD spectroscopy has been extensively used to obtain information on the secondary structures of proteins based on the intrinsic pattern of a spectrum (9). Intrinsic and extrinsic fluorescence have been shown to be capable of characterizing tertiary structures around fluorophores based on the wavelength at the maximum intensity of fluorescence emission and emission intensity (10). Far-UV spectra at 25 and 37 °C showed a similar pattern, with a minimum at ~215 nm and a maximum at ~200 nm, suggesting a mixture of helical and β structures in a folded state at both temperatures (S8A and 10A Figs). Further analyses of the secondary structures indicated that TCan consists of several types of secondary structures, and β structures show a higher proportion than helical structures by ~20% (S8D Fig). These results are in line with the three-dimensional (3D) structure of TCan determined by X-ray crystallography (S9E and F Fig) (9,11): TCan is mainly composed of two antiparallel β-sheets containing 13 β-strands (β1-β13) and four helices (α1-α4) (S10A and D Fig). The 3D structure corresponding to TCan in an intact canavalin (PDB ID:6v7j) is shown in the figure.
Next, three-dimensionally folded structures of TCan were examined using 1-anilino-8-naphthalenesulfonate (ANS) as an extrinsic fluorophore and phenylalanine (Phe) and tyrosine (Tyr) as intrinsic fluorophores (10). ANS is a well-known probe for clarifying partially unfolded structures such as a molten-globule structure, which has the same secondary structure as a native structure without a solid 3D conformation. Thus, binding of ANS to a partially unfolded structure increases its fluorescence emission, that is, a red shift. Although a subtle change was observed, ANS spectra of TCan in the absence and presence of 6 M urea, a chemical denaturant, were similar to the spectrum of the buffer, indicating that ANS did not bind to TCan in either conditions and that TCan has a 3D structure (S9B Fig). The Phe fluorescence spectrum in the absence of urea showed a weak intensity at ~310 nm (S9C Fig), suggesting that the six Phe residues were not exposed to polar solvents and were buried in cores, as indicated in the 3D structure (S9E Fig). The similar spectra at 0 and 6 M urea implied that physicochemical environments around Phe residues are not remarkably different in the folded and urea-induced unfolded states. Similarly, the Tyr fluorescence spectrum without urea exhibited a weak maximum emission at ~310 nm (S9D Fig)-similar to that with 6 M urea-demonstrating that five Tyr residues are buried in the cores (S9F Fig) and are located in a similar environment, regardless of urea. To explore the internal dynamic structures and core regions, we examined X-ray B-factors and the hydrophobicity of each residue in TCan (S10 Fig). Higher values of the B-factor have been interpreted as higher flexibility of atoms and/or residues, and residues with higher hydrophobicity are often buried in the core regions of folded proteins (9,12). Most B-factors ranged between 40 and 60 Å 2 , and β-structures showed a tendency of lower B-factors than those of helical structures (S10A Fig). Random coil-like structures, including both terminal parts and loops between structured secondary structures, exhibited high B-factors. The loop region between the two β-sheets also showed a high number of B-factors. In contrast, N-(~β1-~α1) and C-terminal parts (~β12-~β13) and the loop region between the two β-sheets showed low hydropathy scores, whereas the two central regions, ~β4-~β 7 and ~β8-~β11, showed high hydropathy scores (S10B and D Fig). Interestingly, these two regions individually consisted of a β-sheet and displayed low B-factors, revealing a hydrophobic core of TCan (S10D Fig). Considering these results together, we can conclude that TCan, a truncated form of canavalin, has a secondary and tertiary structure similar to the native structure of the globular protein. Hydrophobic cores of the two β-sheets with low flexibility are capped by two helical regions (~α1 in the N-terminal part and ~α2 ~α 4 in the C-terminal part) and most likely contribute to holding a stereo-structure.

Characterization of conformational stability of TCan
To utilize TCan as a biomaterial and diagnostic agent, it is vital to confirm conformational stability. Therefore, we examined the thermal stability of TCan using spectroscopy and calorimetry. Far-UV CD spectra did not change significantly in the temperature range between 10 and ~70 °C; however, at temperatures over ~70 °C, there were marked changes in the spectra (S8A Fig). The disappearance of a positive CD signal at ~200 nm and a simultaneous increase in the magnitude of the negative CD intensity at ~205 nm suggested the disruption of secondary structures, which is often accompanied by global or sub-global unfolding of 3D structures. Tracing of the CD intensity at 204 nm with an increase in temperature manifested a clear transition of heat-induced unfolding of TCan (S8B Fig). The CD intensity decreased from ~70 °C due to thermal denaturation and was saturated at ~80 °C, which indicated that all TCan thermally unfolded. A sharp transition was detected, indicating cooperative unfolding due to the high packing density interior of TCan, which comes from its high hydrophobicity and low flexibility. This revealed that TCan has a 3D structure as a stable globular protein.
In accordance with the CD results, differential scanning calorimetry (DSC) displayed a positive DSC peak in the thermogram, revealing thermal unfolding of TCan by uptake of heat for denaturation (S9C Fig). An endothermic DSC peak appeared from ~70 °C and was at the level of the baseline from ~70 °C. Temperature-dependent changes in the secondary structure suggested that decreases in the content of β-structures and increases in random coil conformations were followed by thermal unfolding of TCan (S8D Fig). We believe that the hydrophobic cores of β-sheets essential for structural stability were disrupted due to heat stress. With the assumption of a reversible reaction, we further carried out quantitative analyses of the conformational stability of TCan based on two-state unfolding. The melting temperature ™, at which unfolded and folded populations of TCan were identical, and the change in enthalpy (ΔH) for unfolding were obtained using CD ( CD ΔH) and DSC ( DSC ΔH) (S6 and 7 Tables). Tm values calculated from CD ( CD Tm) and DSC ( DSC Tm) were ~75 and ~76 °C, respectively, revealing a relatively high Tm as a number of proteins showed Tm lower than 70 °C (13). CD analyses with the best fit also produced an endothermic CD ΔH of ~100 kcal/mol and a change in heat capacity (ΔCp) of ~2 kcal/mol (S6 Table), which falls into the range of values of globular proteins (14). Hydration of the hydrophobic cores would cause a positive ΔCp value. ΔH obtained using the van't Hoff method and DSC thermogram ( DSC ΔHVH) was ~160 kcal/mol (S7 Table), which was higher than that from CD; however, it was still in the range of ΔH for globular proteins. The reversibility of the thermal unfolding of TCan was tested using several approaches (S11 Fig). The far-UV CD spectrum after heat scanning was not superimposed, and a spectrum indicating an unfolded structure remained (S11A Fig). The ANS spectrum of heat-treated TCan manifested an increased fluorescence emission with a red shift, indicative of the exposure of hydrophobic regions of TCan with partial unfolding (S11B Fig). The intrinsic fluorescence spectra of Phe (S11C Fig) and Tyr (S11D Fig) of TCan subjected to heat treatment did not exhibit appreciable changes. The distribution of the hydrodynamic radius (Rh) of TCan after heat incubation was wider than that before heat scanning (S11E Fig). The average Rh was 3.76 nm; however, new molecular species with higher and lower molecular weights appeared to form. A decrease in the light scattering intensity also signified different structural states of TCan depending on the thermal scan (S11F Fig). These results indicate that thermally unfolded TCan cannot refold to its original structure; that is, unfolding of TCan is irreversible. In summary, a systematic investigation revealed the conformational stability of TCan with detailed thermodynamic properties. Although thermal unfolding of TCan is irreversible, it has been demonstrated that TCan with well-packed hydrophobic cores and low flexibility is a fully stable protein for biological and diagnostic applications.

Investigation on oligomeric states of TCan
The association states of proteins are important for understanding the underlying mechanism of the stability and intermolecular recognition of proteins for functional, biological, and diagnostic applications. In this regard, we focused on the colloidal and association states of TCan. Light scattering has been used to examine the association and colloidal states of proteins. The scattering intensity of TCan is larger than that of the buffer (S8E Fig), suggesting that TCan may exist as a multimer. Dynamic light scattering (DLS) revealed a monodisperse Rh with an average Rh of 3.95 nm (S8F Fig). Based on the relationship between the number of residues, protein volume, and Rh (15), it was calculated that approximately six TCan monomers form the oligomers. Size exclusion chromatography with inline multi-angle light scattering (SEC-MALS) also elucidated the oligomeric states of TCan (S8G Fig). Considering that the molecular weight of the TCan monomer is 21,100, the molecular weight of ~140,000 obtained using SEC-MALS indicated that approximately seven Tcan monomers interact intermolecularly to form an oligomer. Furthermore, consistent with DLS and MALS analyses, DSC also showed the oligomeric states of Tcan (S7 Table). We used the fact that the ratio of DSC ΔHVH / DSC ΔHcal can be a criterion for the oligomeric state of proteins (16). As the ratio of DSC ΔHVH (158.0 ± 0.7) / DSC ΔHcal (19.3 ± 0.1) was 8.3 ± 0.1, it was conceived that a Tcan oligomer accommodates approximately eight monomers. Taken together, TCan forms an oligomer containing ~6 to ~8 monomers under physiological conditions. We rationalized that Tcan efficiently uses spontaneous oligomerization by escaping misfolding and aggregation which inactivates TCan. Furthermore, because intermolecular associations such as dimerization and a higher order of oligomerization often strengthen conformational stability (17), oligomer formation contributes to the relatively high stability of TCan against external stresses in addition to internal stabilizing factors, as mentioned above. Oligomerization of Tcan may also be beneficial to protect itself from external perturbations, such as proteolysis, and be functional.

Intermolecular interactions between Tcan and spike proteins
The binding capability of Tcan for the two SARS-CoV-2 spike proteins, S1 and S2, was investigated using isothermal titration calorimetry (ITC). ITC is one of the most powerful and accurate methodologies for studying intermolecular interactions without labeling or immobilization (18)(19)(20). It can elegantly clarify both weak and strong intermolecular interactions by detecting even a small amount of reaction heat. Titration of Tcan to either S1 (S5D Fig, left panel) or S2 (S5D Fig, right panel) produced energetically favorable exothermic heat, revealing interprotein interactions between TCan and spike proteins. The reaction heat of TCan-S1 was greater than that of TCan-S2, and, thus, the magnitude of the negative enthalpy change for binding ( ITC ΔHbind) of TCan-S1 (~-6-~-7 kcal/mol) seemed to be larger than the ITC ΔHbind of TCan-S2 (~-0·8 kcal/mol). The two binding systems did not show saturation at a molar ratio of 3, indicating that intermolecular interactions are not strong. Although binding isotherms were not saturated, we attempted to evaluate the apparent dissociation constant (Kd). The apparent Kd values for the TCan-S1 and TCan-S2 interactions were in the range of ~20-~100 µM and ~10-~80 µM, respectively. These findings suggest that TCan moderately binds to S1 and S2 with similar affinity. Given the similar interprotein affinity, TCan may bind and recognize a spike protein in a different thermodynamic way: the TCan-S1 interaction is more enthalpically driven, whereas entropy is a predominant player in the TCan-S2 interaction. It should be noted that ITC results, in addition to information on oligomeric states (S5D Fig and S7 Table), collaborate with the Cryo-EM and TEM image (Fig 2A-D and S4 Fig) which demonstrated binding of several TCan, that is, TCan oligomers, to the surfaces of HCoV-229E and SARS-CoV-2 through S1 and S2.

Mitogenic response induced by Con A and BE in mice splenocytes
Con A is well-known for inducing the mitogenic response in splenocytes that activate the immune system, recruit lymphocytes, and elicit cytokine production (21). To compare the mitogenic effects induced by Con A and BE, we investigated T helper (Th) cytokine (IFN- for Th1, IL-17 for Th17, and IL-4, IL-5, and IL-13 for Th2 cells) production by treating splenocytes with Con A or BE at a concentration of 2.5 μg/mL (S12 Fig). The results showed that compared to BE, Con A induced significantly higher production of all Th cytokines. Although production of IFN-, IL-17, IL-5 and IL-13 was increased in BE-treated splenocytes over the duration of the experiment, the production was significantly lower than that in the Con A-treated group.

Effects of Con A and BE on survival rate of mice
To examine and compare the toxic effects of Con A and BE in a living system, we primarily investigated the effects of Con A and BE on mortality after intratracheal instillation in C57BL/6 mice (S13 Fig). The survival rate was monitored every hour in C57BL/6 mice divided into seven groups: vehicle control (VC), Con A-treated at the concentration of 40, 80 and 160 mg/kg body weight (BW) (Con A 40, Con A 80, Con A 160 groups), and BEtreated at the concentration of 75, 160 and 200 mg/kg BW (BE 75, BE 160, BE 200 groups) for 24 h. The VC and BE-treated groups exhibited a survival rate of 100%, while the Con A-treated groups exhibited a decrease in survival. The survival rate for the mice in the Con A-treated groups began to decrease from the 17 th h of treatment, and no survival of mice in the Con A 160 group was observed by the 24 th h of treatment. Meanwhile, 60% survival was observed in mice in the Con A 40 and Con A 80 groups after 24 h of treatment. These results indicate that BE was not toxic to mice, which was consistent with the in vitro cytotoxicity results.
Effects of Con A and BE on liver toxicity of mice Con A can activate T cells to secrete cytokines that cause liver injury (22). To compare the effects of Con A and BE on liver injury, the levels of serum AST, ALT, TBIL, and GGT, which are the biomarkers of liver function, in mice were evaluated at 24 h after Con A and BE administration (S14 Fig). The result showed that the mode of administration impacted hepatic biomarker levels. Although, administration by intratracheal instillation of both Con A and BE-treated mice did not significantly affect the level of hepatic biomarkers, the high concentration of Con A (15 mg/kg BW) administered intravenously remarkably increased the expression of (p value < .05) all biomarkers.

Effects of Con A and BE on lung injury of mice
Animal experiments were conducted to compare lung toxicity by direct respiratory exposure to Con A and BE. The following parameters were analyzed to assess lung injury: (i) lung and spleen organ weight; (ii) histological analysis of lung and spleen tissue; and (iii) differential cell counts and cytokine expression in the BALF. Increasing concentrations of intratracheal Con A instillation gradually increased % lung weight per BW compared with the VC. However, BE did not impact % lung weight compared to VC. In fact, increased lung weight indicates a significant increase in pulmonary vascular permeability and inflammatory cell infiltration into damaged lung regions (S15 Fig). This result may be related to the increase in various types of inflammatory cells, including macrophages, neutrophils, and lymphocytes, in the BALF of Con A-instilled groups. In contrast to the lung weight, neither Con A nor BE altered the spleen weight up to doses of 5 mg/kg BW. A slight decrease in the spleen weight of mice was observed at the highest level (10 mg/kg BW) of Con A instillation. Spleen is a source of inflammatory cells that rapidly accumulate at sites of tissue injury. Decreased spleen weight indicates that inflammatory cells in the spleen are rapidly deployed to sites of lung injury. Additionally, changes in the abundance of different inflammatory cell types were assessed in the BALF after Con A or BE instillation in mice to determine the pulmonary inflammatory response. The results revealed a significant increase in the total number of cells, as well as in the counts of macrophages, eosinophils, and neutrophils in the BALF of Con A-instilled mice as compared to the respective VC group (S16 Fig). However, an increase in cell numbers was not observed with increasing concentration of Con A (1-10 mg/kg BW) instillation. In contrast, the abundance of different cell types did not differ in BE-instilled mice compared to the VC. Moreover, lymphocytes were not observed in the BALF of Con A-or BE-instilled mice. Meanwhile, the spleen weight was also not increased by Con A or BE instillation at the tested concentration levels. An increase in spleen weight could indicate the mitotic transformation of lymphocytes from small to large cells to counteract lectin administration, as previously observed in case of plant lectin toxicity (23). In contrast, lung weight was increased in Con A-instilled mice, which may have been caused in response to water accumulation in the lungs after activated pulmonary neutrophil-induced pulmonary permeability. Alternatively, BE may control neutrophil activation in the lung and reduce ROS production, thereby, reducing pulmonary damage (24). The pathological changes in organ and inflammatory cells induced in response to lectins resulted in increased expression of inflammatory cytokines (S17 Fig). Proinflammatory Th1, Th2, and Th17 cytokines play important roles in triggering lung inflammatory responses (25)(26)(27). Therefore, different cytokines, including IL-1β, IFN, and TNFα secreted by Th1, IL-4, IL-5 and IL-13 secreted by Th2, and IL-17 secreted by Th17 cells were measured in the BALF of Con A-or BE-instilled mice. Proinflammatory cytokine levels in BALF were highly increased in the Con A-instilled group (compared to the VC). Th2 cytokine (IL-4, IL-5, and IL-13) levels in the BALF were significantly increased in the Con A-instilled group (compared to the VC); however, the levels of these cytokines appeared to decrease in the Con A-instilled group in a dose-dependent manner. These results are similar to those observed for cellular changes in BAL cells and are more likely to be related to a decrease in eosinophils. However, no change was observed in cytokine levels within the BALF of the BE-instilled group. Consistently, Con A treatment has been reported to induce proinflammatory cytokine expression in human peripheral blood mononuclear cells (28). Meanwhile, griffithsin, which exhibits effects similar to those of BE, does not induce the production of such cytokines (28). Moreover, a range of cytokines were expressed following Con A treatment with the highest expression being of IFN followed by that of IL-17, IL-5, IL-13, TNF-a, IL-4, and IL-1β. A previous study showed that IFN can induce ROS generation and ferroptosis in tumor cells (29). Thus, Con A treatment may induce ferroptosis by upregulating IFN. Moreover, cytokine storms reportedly induce the dysfunction of multiple organs, including the liver, and ultimately can cause death (30). Therefore, high expression of proinflammatory cytokines and pathological changes within organs, such as the liver and lung, following Con A treatment could explain the high mortality observed in these mice. The results further demonstrated that Con A can mimic the conditions observed in autoimmune hepatitis by inducing acute liver injury as indicated by an increase in the levels of hepatic biomarkers and several proinflammatory cytokines (31). Thus, unlike the regular modes of programmed cell death, such as apoptosis and pyroptosis, the observation of cytotoxicity, ROS generation and inflammatory cytokine production, may indicate the occurrence of ferroptosis in Con A-treated cells (32). Indeed, further analysis is required to verify the ferroptosis-related pathological changes in response to Con A treatment.

Effect of Con A and BE on cytotoxicity and ROS generation
Relative cell viability was gradually reduced with increasing concentrations of Con A or BE in all cell lines in a dose-dependent manner (S18 Fig). However, BE induced a lower rate of cytotoxicity. Concordantly, previous studies have shown that Con A is highly cytotoxic, while griffithsin exhibits low cytotoxicity, comparable to that of BE (28). The IC20 of Con A in HepG2, MH-S, A549, and Huh7 cells treated for 6 h was determined to be 22.44, 35.95, 38.05, and 50.15 µg/mL, respectively. The IC20 of BE for HepG2, MH-S, A549, and Huh7 cells treated for 6 h was determined to be 86·95, 167.69, 110.04, and 232.01 µg/mL, respectively. The higher IC20 of BE indicated the lower cytotoxicity of BE (compared to Con A). In addition, different cells lines exhibited different sensitivities to Con A and BE, which may be attributed to the differences in protein expression during exposure to Con A (33). Similar to the cytotoxic effect of Con A, a lower tolerance of HepG2 to the cytotoxic potency of doxorubicin has also been observed previously (33). Reactive oxygen species (ROS) levels in cells in response to Con A and BE treatment were measured using DCFH-DA staining (34). As observed for the cytotoxic effects, ROS production was observed in a concentration-dependent manner in all cell types, including A549, MH-S, HepG2, and Huh7 cells (S17 Fig). A comparably high and low level of ROS was observed in A549 and Huh7 cells, respectively, in response to Con A treatment. Similarly, a previous study reported that Huh7 cells have a high tolerance to hypoxic conditions (33). Consistent with the cytotoxicity assay results, BE treatment resulted in remarkably lower ROS production than Con A.             Figure S14. Changes in the levels of hepatic biomarkers (AST, ALT, TBIL, and GGT) in response to Con A and BE instillation in mice. Biomarker concentrations were measured in the blood serum of Con A-and BEadministered mice (intravenously or intratracheal instillation; 5-15 or 10-40 mg/kg BW, respectively). Data represent mean ± SD (n = 5 mice per group). **** p < .0001 vs. VC. AST, aspartate aminotransferase; ALT, alanine aminotransferase; TBIL, total bilirubin level; GGT, -glutamyl transferase. Figure S15. Changes in lung and spleen weight-to-body weight ratio after Con A and BE instillation in mice. (A) Lung and (B) spleen weights at a dose of 1-10 mg/kg body weight (BW) were calculated with the following formula: relative organ weight = organ weight (g)/final body weight (g) × 100%. Data represent mean ± SD (n = 5 per group). *p < .05, **p < .01, ***p < .001 and ****p < .0001 when compared to the corresponding vehicle control (VC), determined using Dunnett's t-test. Figure S16. Changes in inflammatory cell numbers in bronchoalveolar lavage fluid (BALF) of mice following Con A and BE instillation. (A) total cells, (B) macrophages, (C) eosinophils, and (D) neutrophils were counted in BALF. BALF was collected and analyzed 24 h after vehicle, Con A, or BE instillation. Data represent mean ± SD (n = 5 per group). *p < .05, **p < .01, ***p < .001 and ****p < .0001 compared to the corresponding vehicle control (VC), determined using Dunnett's t-test. BALF) after Con A or BE instillation at doses of 1-10 mg/kg BW in mice. Data represent mean ± SD (n = 5 mice per group). *p < .05, **p < .01, ***p < .001 and ****p < .0001 when compared to the corresponding vehicle control (VC), determined using Dunnett's t-test. IL, interleukin, IFN-γ, interferon gamma, TNF-α, tumor necrosis factor alpha.  Supplementary Tables S1 Table. Evaluation of the diagnostic performance of BG-RT-PCR and BG-Ag-RDTs for patients with COVID- 19    Whether participants formed a consecutive, random or convenience series 6, Supplemental material 1 Test methods 10a Index test, in sufficient detail to allow replication 8-9 10b Reference standard, in sufficient detail to allow replication 8-9 11 Rationale for choosing the reference standard (if alternatives exist) 8-9 12a Definition of and rationale for test positivity cut-offs or result categories of the index test, distinguishing pre-specified from exploratory 8-9 12b Definition of and rationale for test positivity cut-offs or result categories of the reference standard, distinguishing pre-specified from exploratory 8-9 13a Whether clinical information and reference standard results were available to the performers/readers of the index test 8-9, Supplemental material 1 13b Whether clinical information and index test results were available to the assessors of the reference standard 8-9, Supplemental material 1

Analysis 14
Methods for estimating or comparing measures of diagnostic accuracy 8- 10 15 How indeterminate index test or reference standard results were handled 8- 10 16 How missing data on the index test and reference standard were handled 8-10, Figure 1  17 Any analyses of variability in diagnostic accuracy, distinguishing pre-specified from exploratory 7-9

18
Intended sample size and how it was determined 9 RESULTS

Participants
19 Flow of participants, using a diagram Figure 1, 4 in Supplemental material 2 20 Baseline demographic and clinical characteristics of participants Table 1 21a Distribution of severity of disease in those with the target condition 11, Supplemental material 1 21b Distribution of alternative diagnoses in those without the target condition N. A.

22
Time interval and any clinical interventions between index test and reference standard 7-8 Test results 23 Cross tabulation of the index test results (or their distribution) by the results of the reference standard Figure 2 and S1-2, Table 2 and S1-5

24
Estimates of diagnostic accuracy and their precision (such as 95% confidence intervals) 11-13 Table 2 and S1-5 25 Any adverse events from performing the index test or the reference standard N. A.

DISCUSSION 26
Study limitations, including sources of potential bias, statistical uncertainty, and generalisability

18-19
27 Implications for practice, including the intended use and clinical role of the index test  3, 6, 17-19  OTHER  INFORMATION 28 Registration number and name of registry 3 29 Where the full study protocol can be accessed Supplemental material 2 30 Sources of funding and other support; role of funders 20